Recombinant mycobacterium as a vaccine

ABSTRACT

The invention relates to a recombinant  Mycobacterium  cell for use as a vaccine.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. 371 National Phase Entry Applicationfrom PCT/EP2011/073613, filed Dec. 21, 2011, which claims the benefit ofU.S. Patent Application No. 61/425,442, filed Dec. 21, 2010 and61/436,305 filed Jan. 26, 2011, the disclosures of which areincorporated herein in their entirety by reference.

The invention relates to a recombinant Mycobacterium cell for use as avaccine.

Every year, up to 2 million people die from tuberculosis (TB) (1). Theonly available vaccine against TB is Mycobacterium bovis BacillusCalmette-Guérin (BCG), which was used in humans for the first time in1921 (2). To date, 4 billion doses of BCG have been administered,rendering it the most widely used human vaccine worldwide (3). Yet, weare still far from having achieved eradication of TB. BCG vaccinationprevents tuberculous meningitis and miliary TB in infants (4). However,protection against other forms of TB, notably pulmonary TB inadolescents and adults is inconclusive as emphasized by a meta-analysis,which revealed protective efficacies ranging from 0-80% in adults (5).Therefore, new vaccines against TB are urgently needed. Currently, newvaccination strategies against TB in clinical trials include recombinantBCG to replace canonical BCG as well as subunit vaccines andnon-replicating viral vector-based vaccines to booster BCG prime (6)(7).

The identification of immunologic mechanisms underlying protection canfacilitate rational design of novel vaccination strategies for TBprevention. Moreover, this strategy could reveal biomarkers indicativefor protective immunity that could reveal surrogate endpoints ofclinical outcome in clinical TB vaccine efficacy trials, and thus reducetheir duration as well as facilitate testing of larger numbers ofvaccine candidates in parallel trials. Observational studies focusing onnewly infected, healthy contacts of TB patients and on BCG-vaccinatedinfants have been initiated to define such biomarkers (8). Despiteextensive research on the immune response to TB, the fundamentalelements of protective memory have yet to be elucidated. After BCGvaccination, antigen-specific memory CD4 T cells are difficult to detectdue to the paucity of immunodominant antigens. Currently, the mostwidely used biomarkers are based on elevated frequencies of CD4 T cellsproducing IFNγ. Increasing evidence questions the value of IFNγ ascorrelate of protection in TB (9,10). Undoubtedly IFNγ does play acrucial role in defense against MTB (11), but determination of IFNγalone can no longer be considered as a reliable marker of protectiveimmunity.

A recombinant BCG strain expressing a phagolysosomal escape domain isdescribed in WO 99/101496, the content of which is herein incorporatedby reference. The phagolysosomal escape domain enables the strain toescape from the phagosome of infected host cells by perforating themembrane of the phagosome. In order to provide an acidic phagosomal pHfor optimal phagolysosomal escape activity, a urease-deficientrecombinant strain was developed. This strain is disclosed inWO2004/094469, the content of which is herein incorporated.

A recombinant ΔureC Hly⁺ rBCG (rBCG) strain expressingmembrane-perforating listeriolysin (Hly) of Listeria monocytogenes anddevoid of urease C induces superior protection against aerogenicchallenge with MTB as compared to parental BCG (pBCG) in a preclinicalmodel (12). This vaccine construct has successfully proven safety andimmunogenicity in a phase I clinical trial (U.S. 61/384,375), thecontent of which is herein incorporated by reference.

In the present study, it is shown that rBCG and pBCG induce marked Th1immune responses, whilst only rBCG elicits are profound Th17 response inaddition. It was also observed earlier recruitment of antigen-specific Tlymphocytes to the lung upon MTB infection of rBCG-vaccinated mice.These T cells produced abundant Th1 cytokines after restimulation.Superior protective efficacy of rBCG was apparently dependent on IL17.Elevated IL17 production after rBCG, but not pBCG vaccination, was alsodetected in healthy volunteers during a phase I clinical trial. Ourfindings identify a general immunologic pathway as a marker for improvedvaccination strategies against TB and other diseases.

A subject-matter of the present invention is a recombinant Mycobacteriumcell which comprises a recombinant nucleic acid molecule encoding afusion polypeptide comprising:

(a) a domain capable of eliciting an immune response, and

(b) a phagolysosomal escape domain

for use as vaccine for generating a Th17 immune response.

A further aspect of the present invention is a method for generating aTh17 immune response in a subject in need thereof, comprisingadministering to said subject a recombinant nucleic acid moleculeencoding a fusion polypeptide comprising:

(a) a domain capable of eliciting an immune response, and

(b) a phagolysosomal escape domain.

In a preferred embodiment, the vaccine is a live recombinantMycobacterium cell which comprises a recombinant nucleic acid moleculeencoding a fusion polypeptide comprising (a) a domain capable ofeliciting an immune response and (b) a phagolysosomal escape domain. Thedomain capable of eliciting an immune response is preferably animmunogenic peptide or polypeptide from a pathogen or an immunogenicfragment thereof. In a further embodiment the vaccine is a vaccine basedon an inactivated whole pathogen cell or cell fraction.

The Mycobacterium cell is preferably an M. bovis cell, an M.tuberculosis cell, particularly an attenuated M. tuberculosis cell orother Mycobacteria, e.g. M. microti, M. smegmatis, M. canettii, M.marinum or M. fortuitum. More preferably, the cell is a recombinant M.bovis (BCG) cell, particularly a recombinant M. bovis cell from strainDanish subtype Prague (43). In an especially preferred embodiment, thevaccine is a recombinant urease-deficient Mycobacterium cell. In anespecially preferred embodiment the ureC sequence of the Mycobacteriumcell is inactivated (ΔUrec), e.g. by constructing a suicide vectorcontaining a ureC gene disrupted by a selection marker gene,transforming the target cell with the vector and screening for selectionmarker-positive cells having a urease negative phenotype. Mostpreferably, the cell is recombinant BCG strain Danish subtype Praguecharacterized as rBCG ΔUrec::Hly⁺::Hyg⁺.

The domain capable of eliciting an immune response is preferablyselected from immunogenic peptides or polypeptides from M. bovis, M.tuberculosis or M. leprae or from immunogenic fragments thereof having alength of at least 6, preferably at least 8 amino acids, more preferablyat least 9 amino acids and e.g. up to 20 amino acids. Specific examplesfor suitable antigens are Ag85B (p30) from M. tuberculosis, Ag85B(α-antigen) from M. bovis BCG, Ag85A from M. tuberculosis and ESAT-6from M. tuberculosis and fragments thereof. In other embodiments, thedomain capable of eliciting an immune response is selected fromnon-Mycobacterium polypeptides.

The vaccine of the present invention is suitable for generating a Th17immune response in a mammalian, e.g. human subject. The vaccine ispreferably administered to a human subject in a dose of about 1-10×10⁵,preferably about 2-8×10⁵ cells. The vaccine is preferably administeredas a single dose, e.g. by injection. Subcutaneous injection ispreferred. Further it is preferred to administer the vaccine withoutadjuvant.

The vaccine is preferably a vaccine against mycobacterial infections,such as leprosy or tuberculosis, particularly pulmonary mycobacterialinfections, more particularly pulmonary tuberculosis. Further, thevaccine may be a vaccine against bladder cancer, or an autoimmunedisorder, e.g. an autoimmune skin disorder such as neurodermitis orpsoriasis.

The vaccine may be for generating a Th17 immune response in aMycobacterium-naïve subject, e.g. a human who has not been pre-exposedto an immunogenic Mycobacterium challenge or a human who has not beenpreimmunized with a Mycobacterium-based vaccine, e.g. BCG. Examples ofsuch subjects are, e.g., newborns or children, e.g. up to 8 years, e.g.in areas endemic for mycobacterial infections, such as tuberculosis, orpersons at risk in non-endemic areas. Alternatively, the vaccine may beadministered to a subject, e.g. a human who has been pre-exposed to animmunogenic Mycobacterium challenge or a human who has been preimmunizedwith BCG.

In an especially preferred embodiment, the vaccine is used forgenerating a combined Th17 and Th1 immune response.

According to the invention, a Th17 immune response is generated in avaccinated subject. Determination of the Th17 immune response ispreferably carried out in a biological sample derived from said subject,wherein said sample comprises immune cells, particularly T cells and/orNK cells, more particularly antigen-specific T cells such as CD4 Tcells. The sample may be a body fluid or tissue sample, e.g. a blood,serum or plasma sample or a sample from lung or spleen. Methods forcollecting samples are well known in the art.

For a determination of the Th17 immune response it is preferred torestimulate the immune cells present in the sample in the subject to beanalyzed with an immunogen and determining cytokine expression from saidcells. The cells to be analyzed are preferably antigen-specific T cells,more preferably CD4 T cells. The immunogen for the restimulationcorresponds to the immunogen present in the vaccine (the efficacy ofwhich is to be determined). The immunogen may be present either in aform identical to the form present in the vaccine or in a differentform. For example, when the vaccine comprises an immunogenicpolypeptide, the immunogen in the restimulation step may comprise animmunogenic fragment thereof or vice versa. Preferably, the immunogenused for the restimulation step is a purified polypeptide or peptide. Inorder to test the efficacy of tuberculosis vaccines, particularly a livetuberculosis vaccine as described above, the immunogen may beadvantageously a mycobacterial antigen, e.g. selected from PPD “PurifiedProtein Derivative”, which is a glycerol extract of mycobacteria orAg85A and Ag85B, as well as other mycobacterial antigens and immunogenicfragments thereof (such as described above).

Determination of the Th17 response according to the invention maycomprise determining cells associated with the Th17 response, e.g. IL-17producing cells, by means of surface markers and cytokines present inand/or secreted by said cells. Examples of surface markers are CD4, CD8,IL-23R, CCR4 and/or CCR6. Examples of cytokines present in and/orsecreted by such cells are IL-17, IL-21, IL-22, IL-23, IFN-γ andcombinations thereof. Preferably, the cytokine is IL-17. Such cells maybe determined by cytological methods, e.g. by cell sorting techniquesusing immunological detection reagents such as antibodies specific forcell-surface markers and/or cytokines, which may carry a labelling, e.g.a fluorescence group.

More preferably, cells associated with a Th17 immune response are e.g.CD4 T cells producing and optionally secreting IL-17.

In a further embodiment the determination of the Th17 immune responsecomprises determining a cytokine secreted from Th17 immune responseassociated cells, e.g. IL-17. The cytokine may be determined byimmunological methods using appropriate antibodies, e.g. antibodiesdirected against IL-17.

In a preferred embodiment, a combined Th17 and Th1 immune response isgenerated. A Th1 immune response may be determined by determining cellsassociated with the Th1 response by means of surface markers andcytokines present in and/or secreted by said cells. Examples of surfacemarkers are CD4, CD8, CCR5 and CD62low. Examples of cytokines present inand/or secreted by such cells are IFN-γ, IL-22, and IL-23 andcombinations thereof. Such cells may be determined by cytologicalmethods, e.g. by cell sorting techniques using immunological detectionreagents such as antibodies specific for cell surface markers and/orcytokines, which may carry a labelling, e.g. a fluorescence group.

In a further embodiment, determination of the Th1 immune responsecomprises determining a cytokine secreted from Th1 immuneresponse-associated cells, e.g. IFN-γ, IL-22 and/or IL-23. The cytokinemay be determined by immunological methods using appropriate antibodies,e.g. antibodies directed against IFN-γ, IL-22 and/or IL-23.

Preferably, the Th17 and optionally Th1 immune response is determined ata suitable time after vaccination. For example, the immune response maybe determined 20-50 days, particularly 25-35 days after vaccination.

Further, the invention is described in more detail by the followingFigures and Examples.

FIGURE LEGENDS

FIG. 1: MTB burden in WT mice. S.c. immunization protects againstinfection with MTB 90 days p.i. (A). Bacterial burden is comparablebetween vaccinated and non-vaccinated groups day 7 p.i. (B). CFUdetermination in lung and spleen after aerosol infection with 400 CFUMTB. The cardiac lung lobe (approx. 71/10^(th) of the whole organ) orhalf a spleen was homogenized; the remaining material was used for invitro restimulation assays. Statistical significance determined byMann-Whitney test with two-tailed P values. *, P<0.05; **, P<0.01. Dataare representative of three experiments with similar results.

FIG. 2: Superior cytokine induction after rBCG over pBCG vaccination.Responses in lung (A) and spleen (B) 83 days after s.c. vaccination withrBCG or pBCG. A total of 2.5×10⁵ (lung) or 2×10⁶ cells (spleen) wererestimulated with PPD for 20 hours and supernatants analyzed bymultiplex assays. Cytokine concentrations are depicted as means±SEM offour independent experiments with three replicates each. Backgroundcytokine production from medium controls was subtracted. ANOVA andBonferroni Multiple Comparison Test were applied for statisticalanalysis. *, P<0.05; **, P<0.01.

FIG. 3: Vaccination with rBCG accelerates recruitment ofantigen-specific T cells to the lung upon aerosol infection with MTB.Cytokine secretion by lung cells 7 days after aerosol infection with200-400 CFU MTB. A total of 2×10⁵ cells were stimulated with PPD for 20hours and supernatants analyzed by multiplex assay (A). Cytokineconcentrations are depicted as mean±SEM of two independent experimentswith three replicates each. Background cytokine production from mediumcontrols was subtracted. Cells restimulated with PPD for 6 hours in thepresence of Brefeldin A were analyzed by multicolor flow cytometry (B).Frequencies of responding CD4 T cells are depicted as means±SEM of threeindependent experiments with three replicates each. ANOVA and BonferroniMultiple Comparison Test were applied for statistical analysis. *,P<0.05; **, P<0.01; ***, P<0.001.

FIG. 4 Vaccination with rBCG increases PPD-specific responses in thespleen upon aerosol infection with MTB. Cytokine secretion by spleencells 7 days after aerosol infection with 200-400 CFU MTB. A total of2×10⁶ cells were restimulated with PPD for 20 hours and supernatantsanalyzed by multiplex assays (A). Cytokine concentrations are depictedas means±SEM of four independent experiments with three replicates each.Background cytokine production from medium controls was subtracted.Cells restimulated with PPD for 6 hours in the presence of Brefeldin Awere analyzed by multicolor flow cytometry (B). Frequencies ofresponding CD4 T cells are depicted as mean±SEM of three independentexperiments with three replicates each. ANOVA and Bonferroni MultipleComparison Test were applied for statistical analysis. *, P<0.05; **,P<0.01; ***, P<0.001.#

FIG. 5: Vaccination with pBCG or rBCG does not lead to significantchanges in Treg cell populations. Treg cells in the spleen after s.c.immunization with rBCG or pBCG. Black bars represent CD25+FoxP3+ andwhite bars CD25-FoxP3+ cells. Frequencies of CD4 T cells and CD8 Tregcells 83 days after immunization (A and B) as well as 7 days (C and D)or 90 days (E and F) after aerosol infection with 200-400 CFU MTB. Threemice per group, depicted as mean±SEM. Data are representative of threeindependent experiments with similar results.

FIG. 6: Frequencies of cytokine producing CD8 T cells in the lung aftervaccination and subsequent aerosol infection with MTB. Cytokinesecretion 7 days after aerosol infection with 200-400 CFU MTB. Cellsrestimulated with PPD for 6 hours in the presence of Brefeldin A wereanalyzed by multicolor flow cytometry. Frequencies of responding CD8 Tcells are depicted as mean±SEM of two independent experiments with threereplicates each. ANOVA and Bonferroni Multiple Comparison Test wereapplied for statistical analysis.

FIG. 7: Frequencies of cytokine producing CD8 T cells in the spleenafter vaccination and subsequent aerosol infection with MTB. Cytokinesecretion 7 days after aerosol infection with 200-400 CFU MTB. Cellsrestimulated with PPD for 6 hours in the presence of Brefeldin A wereanalyzed by multicolor flow cytometry. Frequencies of responding CD8 Tcells are depicted as mean±SEM of four independent experiments withthree replicates each. ANOVA and Bonferroni Multiple Comparison Testwere applied for statistical analysis. *, P<0.05; **, P<0.01; ***,P<0.001.

FIG. 8: Immune responses in the lung during persistent MTB infection inrBCG-vaccinated mice. Cytokine secretion by lung cells 90 days afteraerosol infection with 200-400 CFU MTB. Cells were restimulated with PPDfor 20 hours and supernatants analyzed by multiplex assays (A). Cytokineconcentrations are depicted as means±SEM of two independent experimentswith three replicates each. Background cytokine production from mediumcontrols was subtracted. Cells restimulated with PPD for 6 hours in thepresence of Brefeldin A were analyzed by multicolor flow cytometry (B).Frequencies of responding CD4 T cells are depicted as mean±SEM of twoindependent experiments with three replicates each. ANOVA and BonferroniMultiple Comparison Test were applied for statistical analysis. *,P<0.05; **, P<0.01.

FIG. 9: Frequencies of cytokine producing CD8 T cells after vaccinationand subsequent aerosol infection with MTB. Cytokine secretion by cellsisolated from the lung 90 days after aerosol infection with 200-400 CFUMTB. Cells restimulated with PPD for 6 hours in the presence ofBrefeldin A were analyzed by multicolor flow cytometry. Frequencies ofresponding CD8 T cells are depicted as mean±SEM of two independentexperiments with three replicates each. ANOVA and Bonferroni MultipleComparison Test were applied for statistical analysis. *, P<0.05.

FIG. 10: Vaccination induces IL22 but not IL21 production. IL21 (A) andIL22 (B) secretion by cells from spleens (2×10⁶ cells) or lungs (2×10⁵cells) of mice 83 days after vaccination and subsequent aerosolinfection with 200-400 CFU MTB. IL21 concentrations measured from threesamples per group, mean±SEM depicted. For IL22 samples from one groupwere pooled. Data are representative of two (day 83 post vaccination)and five (day 7 p.i.) similar experiments. Cells were restimulated withPPD for 20 hours and supernatants analyzed by ELISA.

FIG. 11: rBCG causes increased recruitment of γδT cells and NK cellswithout significantly altering APC populations. Cells recruited to theperitoneal cavity upon administration of 10⁶ CFU of rBCG or pBCG i.p.Analysis of cell populations in peritoneal lavage fluid by flowcytometry. APC recruited to the peritoneum 5 hours (upper panel) and 6days (lower panel) after i.p. administration of rBCG or pBCG (A). ICS ofT cell populations 5 hours after injection (B). Cells were stimulatedwith αCD3/αCD28 antibodies for 18 hours in the presence of Brefeldin A.ICS of T cell populations 6 days after administration (C). Cells wererestimulated with PPD for 18 hours in the presence of Brefeldin A. Datapresented as summary of three independent experiments with five mice pergroup. Horizontal line indicates median. ANOVA and Bonferroni MultipleComparison Test were applied for statistical analysis. *, P<0.05; **,P<0.01. Cytokines and chemokines detected in peritoneal lavage fluidanalyzed by multiplex assay (D) depicted as mean concentrations±SEM.

FIG. 12: rBCG induces IL17 in human PBMCs from healthy volunteers of aphase I clinical trial. IL17 production by human PBMCs isolated fromhealthy human volunteers of a phase I clinical study. A total of 5×10⁵cells isolated 29 days post vaccination with rBCG or pBCG, wererestimulated with PPD for 20 hours and supernatants analyzed bymultiplex assay. Statistical significance determined by Mann-Whitneytest with one-tailed P value and Welch's correction. *, P<0.05; n=3 forpBCG and n=7 for rBCG.

EXAMPLE Materials and Methods

Mice

Female BALB/c mice were bred at the Bundesinstitut für Risikobewertung(BfR) in Berlin. Mice were 6-8 weeks of age at the beginning of theexperiments and kept under specific pathogen-free (SPF) conditions.Animal experiments were conducted with the approval of the Landesamt fürGesundheit and Soziales (LAGeSo, Berlin, Germany).

Bacteria

The MTB H37Rv and the pBCG and rBCG strains used were describedpreviously (12). Bacteria were grown in Middlebrook 7H9 brothsupplemented with glycerol, 0.05% Tween 80 and ADC. Mid-logarithmiccultures were harvested and stored at −80° C. until use. All stocks weretitrated prior to use. Single cell bacterial suspensions were obtainedby repeated transfer through a syringe with a 27 G needle.

Vaccination and MTB Infection

pBCG or rBCG (10⁶ CFU) were administered subcutaneously (s.c.) in closeproximity to the tail base. Aerogenic infection of mice with MTB wasperformed using a Glas-Col inhalation exposure system. Bacterial burdenswere assessed by mechanical disruption of aseptically removed organs inPBS 0.5% v/v Tween 80 and plating serial dilutions onto Middlebrook 7H11agar plates supplemented with OADC. After 3 weeks, MTB colonies werecounted. Statistical significance of results was determined byMann-Whitney test with two-tailed p-values for non-parametric data usingGraphPad Prism 5.0.

Cell Isolation, Stimulations and Flow Cytometry

Cells were purified as previously described (42). Experimental groupscomprised five mice. Two spleens or lungs were pooled and one sampleprocessed individually, resulting in three samples per group of fivemice for subsequent stimulations. Cells were stimulated with 50 μg/mlPPD (SSI, Copenhagen, Denmark) for 20 hours for cytokine analysis bymultiplex assay or for 6 hours in the presence of 25 μg/ml Brefeldin Afor intracellular cytokine staining (ICS). The following antibodies wereused: CD4 (RM4-5), IFNγ (XMG-1.2), IL2 (JES6-5H4), Ly6G/C (RB6-8C5),CD11b (M1/70), γδ-TCR (GL-3), FoxP3 staining set and CD49b (cloneDX5)all eBioscience. CD8α (YTS169), TNF-α (XT22), CD16/CD32 (2.4G2), F4/80(CI:A3-1) and CD11c (N418) were purified from hybridoma supernatants andfluorescently labeled in house. IL-17 (TC11-18H10) was obtained from BDBiosciences. Cells were analyzed using a FACSCanto II or LSRII flowcytometer and FACSDiva software (BD Biosciences). Cytokines weremeasured using the Bio-Plex Mouse Th1/Th2, IL17 and IL6 bead-basedimmunoassays from Bio-Rad according to manufacturer's instructions. IL21and IL22 were measured by ELISA from R&D systems. Human IL17 wasdetected with a Milliplex 42-plex assay from Millipore.

Peritoneal Lavage

pBCG or rBCG were freshly prepared from mid-logarithmic cultures.Bacteria were washed three times with PBS and concentration determinedby measuring optical density at 580 nm. Administration of 10⁶ CFU wasperformed intraperitoneally. Recruited cells were obtained from theperitoneal cavity 5 hours or 6 days later by injection of 5 ml PBS andanalyzed by flow cytometry. Cytokines and chemokines in lavage fluidwere determined using the Bio-Plex Mouse Cytokine 23-plex kit fromBio-Rad.

Results

Th1/Th17 Responses after rBCG and pBCG Vaccination

In an attempt to elucidate immune mechanisms relevant to TB vaccineefficacy, we compared immune responses to rBCG and pBCG in mice.Superior protective efficacy of rBCG had been originally determinedafter i.v. immunization (12). Here we show that s.c. administration ofrBCG induced comparable levels of protection and retained its superiorefficacy over pBCG (FIG. 1A). Next, we analyzed long-term memoryresponses in lungs and spleens of mice 83 days after s.c. vaccinationwith rBCG or pBCG. Cells were restimulated with PPD and supernatantsanalyzed by multiplex assays for cytokines. Immunization with rBCGinduced significantly higher cytokine production by cells isolated fromthe lung as compared to pBCG (FIG. 2A). These included IFNγ, IL2, IL6,and GM-CSF. In contrast, type 2 cytokines IL4, IL5 and IL10 were notincreased above background levels (data not shown). Intriguingly,approximately 3-fold higher IL17 concentrations were produced by lungcells from rBCG-vaccinated mice. Note that only few cells could beisolated from the lungs of uninfected mice. Consequently, overallcytokine concentrations were lower than in spleen where 10-fold highercell densities per well could be used for stimulation (FIG. 2B). Inspleens, both vaccines elicited equally strong Th1 responses asreflected by comparable concentrations of IL2, IL6, IFNγ and GM-CSF.Yet, spleen cells from rBCG-vaccinated mice produced significantly moreIL17 upon restimulation with PPD as compared to pBCG-vaccinated animals.Thus, immunization with rBCG, but not pBCG, induced concomitant andstrong Th1 and Th17 responses in lungs and spleens, which were sustainedfor prolonged periods of time.

Accelerated Recruitment of MTB-Specific T Cells Upon Infection withVirulent MTB in rBCG-Vaccinated Mice

Th17 cells have been linked to improved immune surveillance (13). Wecompared antigen-specific T cell responses in vaccinated animals uponaerosol infection with virulent MTB, in lungs and spleens 7 days postinfection. Marked IFNγ, IL17, IL2 and GM-CSF production by lung cellsfrom rBCG-vaccinated mice was detected 20 hours after restimulation withPPD (FIG. 3A) or Ag85A peptides (data not shown). In contrast, thesecytokines were barely secreted by cells from pBCG-vaccinated mice. Innon-vaccinated animals infected with MTB, cytokine production was belowdetection limit, in agreement with previous reports that MTB-specific Tcells do not appear before 3 weeks after MTB infection (13). The type 2cytokines IL4, IL5 and IL10 were not detected and TNFα, IL12p70 and IL6were only barely above background levels at this early timepoint postMTB challenge (data not shown).

We determined cytokine production by lung T cells by flow cytometry 7days after MTB infection (FIG. 3B). Cells were stimulated with PPD for 6hours followed by intracellular cytokine staining for IL2, IL17, IFNγand TNFα. In non-vaccinated controls a small proportion of CD4 T cellsproduced IL2, TNFα or IFNγ. In vaccinated mice, CD4 T cells secretedIL2, IFNγ, TNFα and also IL17. Frequencies of single cytokine producingcells were highest, albeit not significant, in the rBCG group. Invaccinated animals we detected multifunctional T cells implicated inprotective immunity (14). Multiple cytokine-producing T cells werepredominantly IL2⁺TNFα⁺ double producers and significantly increasedupon rBCG vaccination. Triple-producer cells were almost exclusivelyIL2⁺IFNγ⁺TNFα⁺ and slightly increased in rBCG compared topBCG-vaccinated mice. Also, IL2⁺TNFα⁺IFNγ⁺IL17⁺ quadruple-positive cellsexclusively appeared in the rBCG group albeit at very low frequencies.

In principle, splenic T cells produced similar cytokine patterns aspulmonary T cells (FIG. 4). IL2 and GM-CSF production was significantlyhigher in the rBCG-vaccinated animals as compared to the pBCG group andbelow detection level in non-vaccinated controls, even though innon-vaccinated controls, a small percentage of CD4 T cells producedIFNγ. In sum, vaccination with either pBCG or rBCG induced CD4 T cellssecreting IFNγ and TNFα with higher frequencies of single andmulti-producers in rBCG-vaccinated mice as compared to the pBCG group.

At 7 days p.i. CD4 T cells were the main cytokine producers; CD8cytokine producers were detected with lower frequencies in the lung(FIG. 6) and spleen (FIG. 7) albeit with similar patterns. It is of notethat significantly higher frequencies of TNFα-single producing CD8 Tcells were detected in the rBCG group.

Differential cytokine production and frequencies of producer cells werenot due to different bacterial burdens at this early timepoint afterinfection, as confirmed by comparable colony forming unit (CFU) numbersin lungs and spleens (FIG. 1B). Total numbers of Treg cells increasedupon infection to a comparable degree in the two vaccinated groups (FIG.5).

Vaccination with rBCG Confers Potent Immune Responses During PersistentInfection

We analyzed MTB-specific immune responses 90 days p.i. when lungbacterial burdens were approximately 10-fold lower in rBCG-immunizedanimals as compared to the pBCG group and 100-fold lower as compared tothe non-vaccinated control group. Cells from lungs of vaccinated miceand untreated controls were restimulated with PPD for 20 hours andcytokine concentrations measured by multiplex assays (FIG. 8A).Cytokines detected upon restimulation were predominantly of type 1.However, we could not detect differences in IFNγ or IL17 between thevaccinated groups during persistent infection. In contrast, amounts ofIL2, IL6, GM-CSF and TNFα were higher in rBCG-vaccinated mice. In allgroups, IL4 and IL5 were below detection limit and some IL12p70 and IL10were measured (data not shown). Additionally, analysis of lung cells bymulticolor flow cytometry revealed predominantly cytokine-producing CD4T cells during persistent infection (FIG. 8B). CD4 T cells secretingonly IFNγ were detected in all groups with similar frequencies. Uponvaccination, CD4 T cells producing IL2, IFNγ, TNFα or IL17 in differentcombinations could be detected as well. Intriguingly, frequencies ofresponding CD4 T cells did not differ significantly between rBCG andpBCG vaccination despite higher concentrations of IL2 and TNFα insupernatants. We assume that both vaccines increased frequencies ofantigen-specific CD4 T cells in the lung during persistent MTB infectionwith rBCG-induced T cells becoming more potent cytokine producers. CD8 Tcells almost exclusively secreted IFNγ with comparable frequencies inall groups whereas significantly higher single TNFα-producing CD8 Tcells were detected in the rBCG group. Multifunctional CD8 T cellsappeared barely above background (FIG. 9).

Vaccination Causes IL22 but not IL21 Production

Th17 cells can produce additional effector cytokines such as IL21 (15)and IL22 (16). IL22-producing cells have been identified in TB patients,but these seem distinct from IL17-producing cells (17). We did notdetect IL21 after stimulation with PPD of spleen or lung cells fromvaccinated and subsequently MTB-infected mice (FIG. 10A). IL22 wasproduced at elevated concentrations by splenocytes stimulated with PPDfor 20 hours (FIG. 10B) in rBCG-immunized mice but did not furtherincrease early after infection with MTB. IL22 production by lung cellswas only observed in the rBCG-vaccinated group and declined afteraerosol MTB infection.

Intraperitoneal rBCG Causes Increased Recruitment of γδ T Cells and NKCells without Significantly Altering APC Populations

We wanted to define the mechanisms underlying preferential Th17 cellinduction after immunization with rBCG. To this end, rBCG and pBCG wereadministered i.p., immigrant cells isolated from the peritoneal cavity 5hours or 6 days after administration and analyzed by flow cytometry(FIG. 11A). Neutrophils (defined as Gr1^(HI), CD11b^(HI), MHCII⁻,CD11c⁻) rapidly entered the peritoneal cavity and remained elevated atday 6 p.i. in pBCG and rBCG groups to the same extent. Frequencies ofresident peritoneal macrophages (defined as CD11b^(HI), F4/80^(HI),Gr1⁻, MHCII^(HI), CD11c⁻) and dendritic cells (CD11c⁺, CD11b^(LO), Gr1⁻)remained unchanged at low percentages. In sum, no significantdifferences were detectable between rBCG and pBCG groups. Peritonealcells harvested 5 hours after vaccination were subjected to polyclonalstimulation (FIG. 11B) with αCD3/αCD28 antibodies. Frequencies of CD4 Tcells and NK cells were comparable between all groups as were IFNγ andIL17 production. Six days after administration, these cells increasednumerically and reached highest frequencies in the rBCG group (FIG.11C). PPD was used for re-stimulation of cells at the 6 day timepoint.CD4 T cells did not produce appreciable amounts of IFNγ or IL17, whilsta substantial proportion of NK cells produced IFNγ after rBCGadministration. γδ T cells have been identified as a major source ofearly IL17 in TB (18). Increased, albeit not significant, proportions ofγδ T cells producing IFNγ were identified 5 hours post-injection withrBCG. This cell population further increased and markedly higherfrequencies of γδ T cells producing IL17 were detected in the rBCG group6 days after injection. CD8 T cells were not detected in the peritoneumin any of the groups. We analyzed cytokines and chemokines present inthe peritoneal cavity by multiplex assay of peritoneal lavage fluid(FIG. 11D). MCP-1, MIP1α, G-CSF and KC were rapidly increased uponinjection; levels of Eotaxin remained unchanged and IL12p40 was detectedin higher concentrations after 6 days. IL12p70, IFNγ, IL1a, IL2, IL3,IL4, IL5, IL10, IL17, GM-CSF, and TNFα concentrations were belowdetection limit whereas IL1β, IL6, IL9, IL13, MIP1β and RANTES could bemeasured but production was comparable between rBCG and pBCG. Thus, rBCGand pBCG induced recruitment of APC as well as chemokine and cytokineproduction at the site of administration to a similar extent.Intriguingly, proportions of γδ T cells secreting IL17 and NK cellsproducing IFNγ were most abundant after rBCG administration.

Vaccination with rBCG Generates IL17-Producing Cells in Humans

Last, we analyzed PBMCs from healthy human volunteers of a phase Iclinical trial to interrogate whether IL17 production was increased inrBCG-vaccinated study participants. Blood from volunteers was taken 29days after immunization with rBCG or pBCG and PBMCs isolated and frozen.PBMCs were thawed and rested over night, followed by 20-hourrestimulation with PPD. Cytokine production was analyzed by multiplexassays. IL17 production was exclusively detected in PBMCs from studyparticipants immunized with rBCG (FIG. 12). Note that a limited numberof samples was available.

Discussion

The identification of immune markers of protection is crucial forrational design of novel TB vaccines. These markers could also establishthe basis for definition of surrogate markers to predict endpoints ofclinical outcome in TB vaccine efficacy trials and thus provideguidelines for improvement of current vaccine candidates. The importanceof key cytokines which activate macrophage antimycobacterial capacitiesincluding IFNγ (21) and TNFα (22), and the necessity for IL2 in theexpansion of memory cells (23) are well established and thus commonlyused to monitor TB vaccine trials.

In an attempt to identify biomarkers of vaccine efficacy, we comparedlong-term memory immune responses elicited by rBCG proven to confersuperior protection over its parental strain, pBCG. Responses differedin both quantitative and qualitative terms. We detected increasedabundance of type 1 cytokines as well as IL17 following vaccination withrBCG in the lung (FIG. 2A). Analysis of vaccine-induced immune responsesin lung is obviously not feasible in the context of clinical trials.Therefore, we also analyzed systemic long-term memory responses (FIG.2B). Intriguingly, comparable concentrations of type-1 directingcytokines were detected in the pBCG and rBCG groups. In contrast, IL17production by splenocytes was significantly elevated upon rBCGvaccination. Thus, we conclude that IL17, rather than IFNγ or IL2qualifies as a marker of superior protection induced by rBCG.

Th17 cells contribute to antimicrobial defense by attracting andactivating neutrophils (24) which are among the first cells to berecruited in response to IL17. It has been shown that IL17 isdispensable during primary MTB infection (25,26), but gains importancein memory responses (13). In addition, recent reports on the expressionof CCR6 on human Th17 cells (27,28) point to a positive feedback loop,because CCR6 is the receptor for CCL20 produced by neutrophils (29) andCCL20-CCR6 has been implicated in immunopathogenisis of TB (30). Thus,Th17 cells could facilitate accelerated recruitment of antigen-specificmemory T cells to the sites of bacterial residence. By analyzingvaccine-induced immune responses 7 days after aerosol infection withMTB, we show that vaccination with rBCG indeed lead to acceleratedrecruitment of effector cells to the sites of bacterial replication. Weobserved increased frequencies of antigen-specific CD4 T cells andelevated production of IL2, IL17, IFNγ and GM-CSF by lung (FIG. 3) andspleen cells (FIG. 4).

Multifunctional CD4 T cells co-producing IL2, IFNγ and TNFα were firstimplicated in successful vaccination strategies against Leishmania major(14) and later also against MTB (31). Recently, these polyfunctional CD4T cells were also detected in clinical TB vaccine trials (32,33). Wedetected polyfunctional CD4 T cells upon rBCG and pBCG vaccination andsubsequent infection (FIGS. 3 and 4); however the composition ofcytokines (IL2, IL17, IFNγ and TNFα) in double- and triple-producersvaried considerably between experiments as well as individual animals.If multifunctional cells were a true correlate of protection, then theiroverall frequencies, which were higher in the rBCG group, rather thantheir composition, seem most relevant.

Why did rBCG induce a Th17 response? Immunization with rBCG and pBCGcaused recruitment of APC as well as chemokine and cytokine producers toa similar extent. Intriguingly, proportions of γδ T cells secreting IL17and NK cells producing IFNγ were highly abundant after rBCG vaccination.This is consistent with reports showing that IL17 can be rapidlyproduced by γδ T cells (34, 18) as well as NKT cells (35). NK cells,which were also increased upon vaccination with rBCG, are an importantsource of early IFNγ. We have already shown that different molecularcomponents released from rBCG reside in the cytosol of infectedmacrophages (12). Nod-2 is an important cytosolic PRR and its engagementhas been linked to the development of Th17 memory T cell responses (19).

Apoptosis induced during bacterial infection induces Th17 cells (36). Wehave obtained evidence that rBCG induces increased apoptosis compared topBCG (12), which could further contribute to increased development ofTh17 cells. Activation of inflammasomes could also contribute to Th17memory responses via production of IL1β (37). NLRP3 for example has beenfound to sense the presence of listeriolysin through changes in ATPlevels (38). Thus, induction of Th17 cells upon rBCG vaccination mightrequire a complex interplay of intracellular stimuli and increasedapoptosis.

Th17 cells are considered instrumental in inflammatory and autoimmunediseases such as collagen-induced arthritis (39), EAE (40, 39) andallergic airway hypersensitivity (41,39) rather than being beneficialfor successful vaccination. This pathogenic role is usually associatedwith development of a profound IL21-mediated inflammatory response. Wenever detected IL21 after vaccination and subsequent MTB infection abovebackground levels in any experiment (FIG. 10A). In addition, we neverobserved signs for autoimmunity or excessive inflammation at the site ofinjection upon vaccination with rBCG nor in the lung up to 200 days postMTB infection.

In a first attempt to compare data from experimental TB in mice withhuman data, we analyzed cytokine profiles of frozen PBMCs from a phase Iclinical trial with rBCG and pBCG. In a limited number of samples wedetected IL17 production after rBCG, but not pBCG, vaccination. Th17cells have been detected in peripheral blood of MTB-infected humans(17). Recently, IL17-producing CD4 T cells have been reported inadolescents vaccinated with a TB vaccine candidate composed of modifiedvaccinia virus Ankara expressing Ag85A (33). Responses peaked betweendays 7 and 28 post vaccination and declined thereafter. This is in linewith our data showing elevated IL17 production at day 29 postvaccination in the rBCG group (FIG. 12). Thus, it is tempting to proposeIL17 as a correlate of protection in TB vaccine trials.

In summary, we show that vaccination with rBCG leads to preferentialgeneration of Th17 cells, likely dependent on intracellular recognitionof bacterial components by Nod-2. These Th17 cells in turn acceleraterecruitment of antigen-specific T cells to the lung. Ultimately, thiscascade of events results in earlier containment of MTB and hence, tosuperior protection by rBCG as compared to pBCG. We detect IL17production exclusively by PBMC from rBCG-vaccinated volunteers in asuccessfully completed phase I clinical trial. Since IL17 seemsinstrumental for accelerated recruitment of antigen-specific T cells tothe sites of MTB replication, future TB vaccines should be tailored toconcomitantly induce balanced Th1 and Th17 responses.

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The invention claimed is:
 1. A method for generating a Th17 immuneresponse in a subject in need thereof, comprising (i) administering tosaid subject a recombinant urease-deficient Mycobacterium bovis fromstrain Danish subtype Prague (rBCG ΔUrec::Hly+::Hyg+) derived from theparental strain Mycobacterium bovis TMC1025BCG Prague (ATCC 35742) and(ii) determining an elevated Th17 immune response by measuring IL-17production, wherein said rBCG elicits an elevated IL-17 production ascompared with a parental BCG (pBCG) vaccination.
 2. The method of claim1, wherein the rBCG generates a Th17 immune response in a human subject.3. The method of claim 1, wherein the rBCG generates a Th17 immuneresponse in a Mycobacterium-naive subject or a subject pre-exposed toMycobacterium challenge.
 4. The method of claim 1, wherein the rBCGgenerates a Th17 and a Th1 immune response in said subject.